Distribution of cardiomyocyte-selective adeno-associated virus serotype 9 vectors in swine following intracoronary and intravenous infusion.

Limited reports exist regarding adeno-associated virus (AAV) biodistribution in swine. This study assessed biodistribution following antegrade intracoronary and intravenous delivery of two self-complementary serotype 9 AAV (AAV9sc) biologics designed to target signaling in the cardiomyocyte considered important for the development of heart failure. Under the control of a cardiomyocyte-specific promoter, AAV9sc.shmAKAP and AAV9sc.RBD express a small hairpin RNA for the perinuclear scaffold protein muscle A-kinase anchoring protein ß (mAKAPß) and an anchoring disruptor peptide for p90 ribosomal S6 kinase type 3 (RSK3), respectively. Quantitative PCR was used to assess viral genome (vg) delivery and transcript expression in Ossabaw and Yorkshire swine tissues. Myocardial viral delivery was 2-5 × 105 vg/µg genomic DNA (gDNA) for both infusion techniques at a dose ∼1013 vg/kg body wt, demonstrating delivery of ∼1-3 viral particles per cardiac diploid genome. Myocardial RNA levels for each expressed transgene were generally proportional to dose and genomic delivery, and comparable with levels for moderately expressed endogenous genes. Despite significant AAV9sc delivery to other tissues, including the liver, neither biologic induced toxic effects as assessed using functional, structural, and circulating cardiac and systemic markers. These results indicate successful targeted delivery of cardiomyocyte-selective viral vectors in swine without negative side effects, an important step in establishing efficacy in a preclinical experimental setting.

Signalosome-Regulated Serum Response Factor Phosphorylation Determining Myocyte Growth in Width Versus Length as a Therapeutic Target for Heart Failure.

BACKGROUND: Concentric and eccentric cardiac hypertrophy are associated with pressure and volume overload, respectively, in cardiovascular disease both conferring an increased risk of heart failure. These contrasting forms of hypertrophy are characterized by asymmetrical growth of the cardiac myocyte in mainly width or length, respectively. The molecular mechanisms determining myocyte preferential growth in width versus length remain poorly understood. Identification of the mechanisms governing asymmetrical myocyte growth could provide new therapeutic targets for the prevention or treatment of heart failure. METHODS: Primary adult rat ventricular myocytes, adeno-associated virus (AAV)-mediated gene delivery in mice, and human tissue samples were used to define a regulatory pathway controlling pathological myocyte hypertrophy. Chromatin immunoprecipitation assays with sequencing and precision nuclear run-on sequencing were used to define a transcriptional mechanism. RESULTS: We report that asymmetrical cardiac myocyte hypertrophy is modulated by SRF (serum response factor) phosphorylation, constituting an epigenomic switch balancing the growth in width versus length of adult ventricular myocytes in vitro and in vivo. SRF Ser103 phosphorylation is bidirectionally regulated by RSK3 (p90 ribosomal S6 kinase type 3) and PP2A (protein phosphatase 2A) at signalosomes organized by the scaffold protein mAKAPß (muscle A-kinase anchoring protein ß), such that increased SRF phosphorylation activates AP-1 (activator protein-1)-dependent enhancers that direct myocyte growth in width. AAV are used to express in vivo mAKAPß-derived RSK3 and PP2A anchoring disruptor peptides that block the association of the enzymes with the mAKAPß scaffold. Inhibition of RSK3 signaling prevents concentric cardiac remodeling induced by pressure overload, while inhibition of PP2A signaling prevents eccentric cardiac remodeling induced by myocardial infarction, in each case improving cardiac function. SRF Ser103 phosphorylation is significantly decreased in dilated human hearts, supporting the notion that modulation of the mAKAPß-SRF signalosome could be a new therapeutic approach for human heart failure. CONCLUSIONS: We have identified a new molecular switch, namely mAKAPß signalosome-regulated SRF phosphorylation, that controls a transcriptional program responsible for modulating changes in cardiac myocyte morphology that occur secondary to pathological stressors. Complementary AAV-based gene therapies constitute rationally-designed strategies for a new translational modality for heart failure.

Insulin-like growth factor 1 reduces coronary atherosclerosis in pigs with familial hypercholesterolemia.

Although murine models of coronary atherosclerotic disease have been used extensively to determine mechanisms, limited new therapeutic options have emerged. Pigs with familial hypercholesterolemia (FH pigs) develop complex coronary atheromas that are almost identical to human lesions. We reported previously that insulin-like growth factor 1 (IGF-1) reduced aortic atherosclerosis and promoted features of stable plaque in a murine model. We administered human recombinant IGF-1 or saline (control) in atherosclerotic FH pigs for 6 months. IGF-1 decreased relative coronary atheroma in vivo (intravascular ultrasound) and reduced lesion cross-sectional area (postmortem histology). IGF-1 increased plaque's fibrous cap thickness, and reduced necrotic core, macrophage content, and cell apoptosis, consistent with promotion of a stable plaque phenotype. IGF-1 reduced circulating triglycerides, markers of systemic oxidative stress, and CXCL12 chemokine levels. We used spatial transcriptomics (ST) to identify global transcriptome changes in advanced plaque compartments and to obtain mechanistic insights into IGF-1 effects. ST analysis showed that IGF-1 suppressed FOS/FOSB factors and gene expression of MMP9 and CXCL14 in plaque macrophages, suggesting possible involvement of these molecules in IGF-1's effect on atherosclerosis. Thus, IGF-1 reduced coronary plaque burden and promoted features of stable plaque in a pig model, providing support for consideration of clinical trials.

Mechanism-Driven Modeling to Aid Non-invasive Monitoring of Cardiac Function via Ballistocardiography.

Left ventricular (LV) catheterization provides LV pressure-volume (P-V) loops and it represents the gold standard for cardiac function monitoring. This technique, however, is invasive and this limits its applicability in clinical and in-home settings. Ballistocardiography (BCG) is a good candidate for non-invasive cardiac monitoring, as it is based on capturing non-invasively the body motion that results from the blood flowing through the cardiovascular system. This work aims at building a mechanistic connection between changes in the BCG signal, changes in the P-V loops and changes in cardiac function. A mechanism-driven model based on cardiovascular physiology has been used as a virtual laboratory to predict how changes in cardiac function will manifest in the BCG waveform. Specifically, model simulations indicate that a decline in LV contractility results in an increase of the relative timing between the ECG and BCG signal and a decrease in BCG amplitude. The predicted changes have subsequently been observed in measurements on three swine serving as pre-clinical models for pre- and post-myocardial infarction conditions. The reproducibility of BCG measurements has been assessed on repeated, consecutive sessions of data acquisitions on three additional swine. Overall, this study provides experimental evidence supporting the utilization of mechanism-driven mathematical modeling as a guide to interpret changes in the BCG signal on the basis of cardiovascular physiology, thereby advancing the BCG technique as an effective method for non-invasive monitoring of cardiac function.

Low-intensity interval exercise training attenuates coronary vascular dysfunction and preserves Ca²âº-sensitive K⁺ current in miniature swine with LV hypertrophy.

Coronary vascular dysfunction has been observed in several models of heart failure (HF). Recent evidence indicates that exercise training is beneficial for patients with HF, but the precise intensity and underlying mechanisms are unknown. Left ventricular (LV) hypertrophy can play a significant role in the development of HF; therefore, the purpose of this study was to assess the effects of low-intensity interval exercise training on coronary vascular function in sedentary (HF) and exercise trained (HF-TR) aortic-banded miniature swine displaying LV hypertrophy. Six months postsurgery, in vivo coronary vascular responses to endothelin-1 (ET-1) and adenosine were measured in the left anterior descending coronary artery. Baseline and maximal coronary vascular conductance were similar between all groups. ET-1-induced reductions in coronary vascular conductance (P < 0.05) were greater in HF vs. sedentary control and HF-TR groups. Pretreatment with the ET type A (ET(A)) receptor blocker BQ-123 prevented ET-1 hypersensitivity in HF animals. Whole cell voltage clamp was used to characterize composite K(+) currents (I(K(+))) in coronary smooth muscle cells. Raising internal Ca(2+) from 200 to 500 nM increased Ca(2+)-sensitive K(+) current in HF-TR and control, but not HF animals. In conclusion, an ET(A)-receptor-mediated hypersensitivity to ET-1, elevated resting LV wall tension, and decreased coronary smooth muscle cell Ca(2+)-sensitive I(K(+)) was found in sedentary animals with LV hypertrophy. Low-intensity interval exercise training preserved normal coronary vascular function and smooth muscle cell Ca(2+)-sensitive I(K(+)), illustrating a potential mechanism underlying coronary vascular dysfunction in a large-animal model of LV hypertrophy. Our results demonstrate the potential clinical impact of exercise on coronary vascular function in HF patients displaying pathological LV hypertrophy.

Endurance exercise training does not limit coronary atherosclerosis in familial hypercholesterolemic swine.

Human studies demonstrate that physical activity reduces both morbidity and mortality of coronary heart disease (CHD) including decreased progression and/or regression of CHD with life-style modification which includes exercise. However, evidence supporting an intrinsic, direct effect of exercise in attenuating the development of CHD is equivocal. One limitation has been the lack of a large animal model with clinically evident CHD disease. Thus, we examined the role of endurance exercise in CHD development in a swine model of familial hypercholesterolemia (FH) that exhibits robust, complex atherosclerosis. FH swine were randomly assigned to either sedentary (Sed) or exercise trained (Ex) groups. At 10 months of age, Ex pigs began a 10 months, moderate-intensity treadmill-training intervention. At 14 months, all pigs were switched to a high-fat, high-cholesterol diet. CHD was assessed by intravascular ultrasound (IVUS) both prior to and after completion of 6 months on the HFC diet. Prior to HFC diet, Ex resulted in a greater coronary artery size in the proximal and mid sections of the LCX compared to SED, with no effect in the LAD. After 6 months on HFC diet, there was a 5-6 fold increase in absolute plaque volume in all segments of the LCX and LAD in both groups. At 20 months, there was no difference in vessel volume, lumen volume, absolute or relative plaque volume in either the LCX or LAD between Sed and Ex animals. These findings fail to support an independent, direct effect of exercise in limiting CHD progression in familial hypercholesterolemia.

Western Diet-Fed, Aortic-Banded Ossabaw Swine: A Preclinical Model of Cardio-Metabolic Heart Failure.

The development of new treatments for heart failure lack animal models that encompass the increasingly heterogeneous disease profile of this patient population. This report provides evidence supporting the hypothesis that Western Diet-fed, aortic-banded Ossabaw swine display an integrated physiological, morphological, and genetic phenotype evocative of cardio-metabolic heart failure. This new preclinical animal model displays a distinctive constellation of findings that are conceivably useful to extending the understanding of how pre-existing cardio-metabolic syndrome can contribute to developing HF.

Feasibility of a nanomaterial-tissue patch for vascular and cardiac reconstruction.

Vascular and cardiac reconstruction involves the use of biological patches to treat trauma and defects. An in vivo study was performed to determine the remodeling and biologic effects of novel nanostructured vascular patches with and without gold nanoparticles. Porcine vascular tissue was decellularized and conjugated with gold nanoparticles to evaluate if integration would occur while avoiding rupture and stenosis. Swine underwent a bilateral patch angioplasty of the carotid arteries with experimental patches on the right and control patches of bovine pericardium on the left. Animals were sacrificed after surgery and at 3 and 9 weeks. Ultrasound was performed during surgery, every 3 weeks, and before euthanasia. Endothelial regeneration was examined using Evans Blue dye and histology using Trichrome and H&E. There was a 100% success rate of implantation with 0% mortality. All patches were patent on ultrasound. At 3 weeks, experimental patches had regenerating endothelial cell growth and normal healing responses. At 9 weeks, the experimental patches demonstrated excellent integration. Histology demonstrated cellular in-growth into the experimental patches and no major immune reactions. This is one of the first studies to demonstrate the feasibility of nanomaterial-tissue patches for vascular and cardiac reconstruction.

Saxagliptin and Tadalafil Differentially Alter Cyclic Guanosine Monophosphate (cGMP) Signaling and Left Ventricular Function in Aortic-Banded Mini-Swine.

BACKGROUND: Cyclic guanosine monophosphate-protein kinase G-phosphodiesterase 5 signaling may be disturbed in heart failure (HF) with preserved ejection fraction, contributing to cardiac remodeling and dysfunction. The purpose of this study was to manipulate cyclic guanosine monophosphate signaling using the dipeptidyl-peptidase 4 inhibitor saxagliptin and phosphodiesterase 5 inhibitor tadalafil. We hypothesized that preservation of cyclic guanosine monophosphate cGMP signaling would attenuate pathological cardiac remodeling and improve left ventricular (LV) function. METHODS AND RESULTS: We assessed LV hypertrophy and function at the organ and cellular level in aortic-banded pigs. Concentric hypertrophy was equal in all groups, but LV collagen deposition was increased in only HF animals. Prevention of fibrotic remodeling by saxagliptin and tadalafil was correlated with neuropeptide Y plasma levels. Saxagliptin better preserved integrated LV systolic and diastolic function by maintaining normal LV chamber volumes and contractility (end-systolic pressure-volume relationship, preload recruitable SW) while preventing changes to early/late diastolic longitudinal strain rate. Function was similar to the HF group in tadalafil-treated animals including increased LV contractility, reduced chamber volume, and decreased longitudinal, circumferential, and radial mechanics. Saxagliptin and tadalafil prevented a negative cardiomyocyte shortening-frequency relationship observed in HF animals. Saxagliptin increased phosphodiesterase 5 activity while tadalafil increased cyclic guanosine monophosphate levels; however, neither drug increased downstream PKG activity. Early mitochondrial dysfunction, evident as decreased calcium-retention capacity and Complex II-dependent respiratory control, was present in both HF and tadalafil-treated animals. CONCLUSIONS: Both saxagliptin and tadalafil prevented increased LV collagen deposition in a manner related to the attenuation of increased plasma neuropeptide Y levels. Saxagliptin appears superior for treating heart failure with preserved ejection fraction, considering its comprehensive effects on integrated LV systolic and diastolic function.

Ovariectomy increases L-type Ca(2+) channel activity in porcine coronary smooth muscle.

OBJECTIVE: Sex hormone status has been demonstrated to play a role in the regulation of ion channel activity. We previously demonstrated increased L-type Ca channel current (ICa) in the coronary smooth muscle cells (SMCs) of male swine compared with female swine. In male swine, endogenous testosterone increases ICa in SMCs by enhanced expression of the pore-forming α1 subunit Cav1.2. Conversely, the role of sex hormones in female swine has not previously been investigated. Therefore, the purpose of the current study was to determine the effect of ovariectomy (OVX) on L-type Ca channel activity and expression in female Yucatan miniature swine. METHODS: Sexually mature female swine were obtained from a breeder and either left intact (intact female [IF]; n = 5) or ovariectomized (n = 6). RESULTS: Sensitivity to depolarization-induced contractions was increased by OVX. Accordingly, mean (SEM) ICa was enhanced in the OVX group (-9.5 [0.6] pA/pF) compared with the IF group (-4.5 [0.3] pA/pF), although L-type Ca channel α1 subunit (Cav1.2; α1c) messenger RNA (mRNA) and protein expressions were unchanged.Among the L-type Ca channel ß subunits, ß1 (188 [31]) and ß2a (561 [79]) had higher mRNA expression levels (target/18S) than ß3 (9 [1]) and ß4 (2 [0.1]). Although ß2a, ß3, and ß4 mRNA and protein expressions were not different between groups, protein expression of the ß1 subunit (Cavß1) was decreased in the OVX group compared with the IF group. CONCLUSIONS: Endogenous female hormones inhibit L-type Ca channel activity in coronary SMCs potentially via the up-regulation of Cavß1 subunit expression.

Differential effects of androgens on coronary blood flow regulation and arteriolar diameter in intact and castrated swine.

BACKGROUND: Low endogenous testosterone levels have been shown to be a risk factor for the development of cardiovascular disease and cardiovascular benefits associated with testosterone replacement therapy are being advocated; however, the effects of endogenous testosterone levels on acute coronary vasomotor responses to androgen administration are not clear. The objective of this study was to compare the effects of acute androgen administration on in vivo coronary conductance and in vitro coronary microvascular diameter in intact and castrated male swine. METHODS: Pigs received intracoronary infusions of physiologic levels (1-100 nM) of testosterone, the metabolite 5α-dihydrotestosterone, and the epimer epitestosterone while left anterior descending coronary blood flow and mean arterial pressure were continuously monitored. Following sacrifice, coronary arterioles were isolated, cannulated, and exposed to physiologic concentrations (1-100 nM) of testosterone, 5α-dihydrotestosterone, and epitestosterone. To evaluate effects of the androgen receptor on acute androgen dilation responses, real-time PCR and immunohistochemistry for androgen receptor were performed on conduit and resistance coronary vessels. RESULTS: In vivo, testosterone and 5α-dihydrotestosterone produced greater increases in coronary conductance in the intact compared to the castrated males. In vitro, percent maximal dilation of microvessels was similar between intact and castrated males for testosterone and 5α-dihydrotestosterone. In both studies epitestosterone produced significant increases in conductance and microvessel diameter from baseline in the intact males. Androgen receptor mRNA expression and immunohistochemical staining were similar in intact and castrated males. CONCLUSIONS: Acute coronary vascular responses to exogenous androgen administration are increased by endogenous testosterone, an effect unrelated to changes in androgen receptor expression.

Endogenous testosterone attenuates neointima formation after moderate coronary balloon injury in male swine.

AIMS: Previous studies from our laboratory have demonstrated that testosterone increases coronary smooth muscle protein kinase C delta (PKC delta) both in vivo and in vitro and inhibits coronary smooth muscle proliferation by inducing G(0)/G(1) cell cycle arrest in a PKC delta-dependent manner. The purpose of the present study was to determine whether endogenous testosterone limits coronary neointima (NI) formation in a porcine model of post-angioplasty restenosis. METHODS AND RESULTS: Sexually mature, male Yucatan miniature swine were either left intact (IM), castrated (CM), or castrated with testosterone replacement (CMT; Androgel, 10 mg/day). Angioplasty was performed in both the left anterior descending and left circumflex coronary arteries with balloon catheter overinflation to induce either moderate (1.25-1.3 x diameter; 3 x 30 s) or severe (1.4x diameter; 3 x 30 s) injury, and animals were allowed to recover for either 10 or 28 days. Injured coronary sections were dissected, fixed, stained (Verheoff-Van Gieson, Ki67, PKC delta, p27), and analysed. Vessels without internal elastic laminal rupture were excluded. Following moderate injury, intimal area, intima-to-media ratio (I/M), and I/M normalized to rupture index (RI) were increased in CM compared with IM and CMT. RI, medial area, and intimal/medial thickness (IMT) were not different between groups. NI formation was inversely related to serum testosterone concentration. Conversely, following severe injury, there were no significant differences between the groups. Testosterone inhibited proliferation and stimulated PKC delta and p27(kip1) expression during NI formation (10 days post-injury). CONCLUSION: These findings demonstrate that endogenous testosterone limits coronary NI formation in male swine and provides support for a protective role for testosterone in coronary vasculoproliferative diseases, such as restenosis and atherosclerosis.